In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing. This phenomenon occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms.
First observed in the nematode, the posttranscriptional gene silencing defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated as RNA interference (RNAi). This term has come to generally refer to the process of gene silencing involving dsRNA which leads to the sequence-specific reduction of gene expression. It is currently believed that RNAi represents a form of immunity and protection from invasion by exogenous sources of genetic material such as RNA viruses and retrotransposons (Eddy, Nature Reviews Genetics, 2001, 2, 919-929; Silva et al., Trends in Molecular Medicine, 2002, 8, 505-508).
RNA genes were once considered relics of a primordial “RNA world” that was largely replaced by more efficient proteins. More recently, however, it has become clear that non-coding RNA genes produce functional RNA molecules with important roles in regulation of gene expression, developmental timing, viral surveillance, and immunity. Not only the classic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but also small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), tiny non-coding RNAs (tncRNAs) and microRNAs (miRNAs) are now known to act in diverse cellular processes such as chromosome maintenance, gene imprinting, pre-mRNA splicing, guiding RNA modifications, transcriptional regulation, and the control of mRNA translation (Eddy, Nature Reviews Genetics, 2001, 2, 919-929; Kawasaki and Taira, Nature, 2003, 423, 838-842). RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, and DNA elimination (Cerutti, Trends in Genetics, 2003, 19, 39-46; Couzin, Science, 2002, 298, 2296-2297).
RNAi was defined in the nematode, following observations that injections of either an antisense RNA or a sense strand RNA disrupted expression (Guo et al., Cell, 1995, 81, 611-620). Subsequently, Fire et al. injected dsRNA (a mixture of both sense and antisense strands) into C. elegans. Injection of both antisense and sense strands resulted in much more efficient silencing than injection of either the sense or the antisense strands alone. Injection of just a few molecules of dsRNA per cell was sufficient to completely silence the homologous gene's expression. Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm, but also in first generation offspring (Timmons et al., Nature, 1998, 395, 854). Single-stranded RNA oligomers of antisense polarity can also be potent inducers of gene silencing. The authors hypothesize that gene silencing is accomplished by RNA primer extension using the mRNA as template, leading to dsRNA that is subsequently degraded, suggesting that single-stranded RNA oligomers are ultimately responsible for the RNAi phenomenon (Tijsterman et al., Science, 2002, 295, 694-697). Some double stranded RNA molecules mediating RNAi are 21-25 nucleotides in length and are referred to as small interfering RNAs (siRNAs).
An additional class of small non-coding RNAs known as microRNAs (miRNAs) participates in regulation of gene expression. In nematodes, fruit flies, and humans, miRNAs are predicted to function as endogenous posttranscriptional gene regulators. Mature miRNAs originate from long endogenous primary transcripts (pri-miRNAs) that are often hundreds of nucleotides in length (Lee et al., Embo J, 2002, 21, 4663-4670). These pri-miRNAs are processed by a nucleolar enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop, hairpin or foldback precursors) (Lee et al., Nature, 2003, 425, 415-419) which are subsequently exported from the nucleus into the cytoplasm through the action of the nuclear export protein exportin-5 (Bohnsack et al., Rna, 2004, 10, 185-191; Lund et al., Science, 2004, 303, 95-98; Yi et al., Genes Dev., 2003, 17, 3011-3016). Once in the cytoplasm, the pre-miRNA is cleaved by Dicer to yield a double-stranded intermediate, but only one strand of this short-lived intermediate accumulates as the mature miRNA (Bartel, Cell, 2004, 116, 281-297; Grishok et al., Cell, 2001, 106, 23-34; Hutvágner et al., Science, 2001, 293, 834-838).
Naturally occurring miRNAs are characterized by imperfect complementarity to their target sequences. Artificially modified miRNAs with sequences completely complementary to their target RNAs have been designed and found to function as siRNAs that inhibit gene expression by reducing RNA transcript levels. Synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules were demonstrated to target genes for silencing by degradation and not translational repression (McManus et al., RNA, 2002, 8, 842-850). Consequently, miRNAs are believed to primarily direct translation repression, although examples of miRNA-mediated target mRNA degradation have been observed (Yekta et al., Science, 2004, 304, 594-596).
Recently identified miRNA functions include control of cell proliferation, cell death, fat metabolism in flies, neuronal patterning in nematodes, modulation of hematopoietic lineage differentiation in mammals and control of leaf and flower development in plants. Thus, miRNAs participate in a variety of cellular processes and biological functions (Bartel, Cell, 2004, 116, 281-297).
The process of RNAi can be divided into two general steps: the initiation step occurs when the gene silencing trigger (dsRNA) is processed into siRNAs by an RNase III-like dsRNA-specific enzyme known as Dicer, and the effector step, during which the siRNAs are incorporated into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC). RISC is believed to use the siRNA molecules as a guide to identify complementary RNAs, and an endoribonuclease (to date unidentified) cleaves these target RNAs, resulting in their degradation (Cerutti, Trends in Genetics, 2003, 19, 39-46; Grishok et al., Cell, 2001, 106, 23-34).
Like siRNAs, miRNAs are processed by Dicer and are approximately the same length, and possess the characteristic 5′-phosphate and 3′-hydroxyl termini. The miRNAs are also incorporated into a ribonucleoprotein complex, the miRNP, which is similar, if not identical to the RISC (Mourelatos et al., Genes & Development, 2002, 16, 720-728).
Tudor staphylococcal nuclease (Tudor-SN; Epstein Barr virus nuclear antigen-2 coactivator p100; EBNA2 coactivator p100; staphylococcal nuclease domain-containing protein 1; SND1) is a component of the RISC enzyme in C. elegans, Drosophila and mammals. Purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases, and a specific competitive inhibitor of micrococcal nucleases inhibits both RISC and Tudor-SN activity. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain and could thus contribute to the RNA degradation observed in RNAi (Caudy et al., Nature, 2003, 425, 411-414).
Tudor-SN was first identified as a nuclear protein that specifically augmented gene activation by the Epstein-Barr virus nuclear antigen 2 (EBNA 2), a product of one of the first genes expressed by the Epstein-Barr virus (EBV) upon infection of B lymphocytes. This coactivation occurs through the acidic domain of EBNA 2. In addition to binding EBNA 2, Tudor-SN also binds both units of the transcription factor TFIIE, suggesting that it may act as a bridge between EBNA 2 and the basal transcription machinery during EBV infection of B lymphocytes. As shown by the expression of an antisense Tudor-SN RNA, Tudor-SN is essential for cell growth. Additionally, Tudor-SN is capable of binding single-stranded DNA (Tong et al., Mol. Cell. Biol., 1995, 15, 4735-4744).
As further evidence that Tudor-SN functions as a coactivator, Tudor-SN interacts with the signal transducer and activator of transcription 6 (STAT6) both in vivo and in vitro and enhances the STAT6-mediated transcription activation and interleukin-4-induced gene activation in B cells, which is inhibited by overexpression of an antisense Tudor-SN construct. Furthermore, Tudor-SN interacts with the large subunit of RNA polymerase II, suggesting that Tudor-SN functions as a bridge between STAT6 and the basal transcription machinery (Yang et al., Embo J., 2002, 21, 4950-4958).
Tudor-SN was found to interact with an additional member of the signal transducer and activator of transcription, STAT5, both in vivo and in vitro, and this interaction was mediated by both the tudor and staphylococcal nuclease-like domains of Tudor-SN. Prolactin stimulation of mouse mammary epithelial cells increased Tudor-SN protein levels and also enhanced STAT5-dependent transcriptional activation. These data suggest a mechanism for prolactin-induced transcription in which prolactin stabilizes Tudor-SN, which in turn cooperates with STAT5 in transcriptional activation (Paukku et al., Mol. Endocrinol., 2003, 17, 1805-1814).
Tudor-SN was identified as a constituent of endoplasmic reticulum and cytosolic lipid droplets from bovine milk-secreting cells, and was also found in cytosol from bovine and murine lactating mammary gland, in storage lipid droplets from murine adipocytes and in endoplasmic reticulum from murine liver. Since Tudor-SN is on the surface of lipid droplets within the cell, it may play some role in formation or growth of these droplets (Keenan et al., Biochim. Biophys. Acta, 2000, 1523, 84-90). Tudor-SN is localized to both the membrane/organelle fraction and the nuclei of mammary epithelial cells. An increase in Tudor-SN protein is associated with milk production and occurs without a corresponding increase in the abundance of Tudor-SN mRNA, suggesting that the level of Tudor-SN is the result of a post-translational modification. Tudor-SN protein expression is also stimulated in response to lactogenic stimuli in cultured mammary cells (Broadhurst et al., J. Endocrinol., 2001, 171, 329-337). These data indicate that Tudor-SN participates in the molecular mechanisms controlling milk production in mammary epithelial cells.
Analyses of Tudor-SN structure revealed the presence of a single tudor domain, a domain first identified in the Drosophila posterior group gene tudor which encodes a protein required during oogenesis (Ponting, Trends. Biochem. Sci., 1997, 22, 51-52). Tudor-SN contains 4 additional repeats, each of which is homologous to Staphylococcus aureus nuclease (SNase), yet lack the SNase catalytic residues. Based upon structural similarities, the SN-like domains of Tudor-SN may adopt the SN-fold, a structure belonging to the oligonucleotide/oligosaccharide-binding (OB)-fold superfamily which includes a variety of nucleic acid binding proteins (Callebaut et al., Biochem. J., 1997, 321, 125-132; Ponting, Protein Sci., 1997, 6, 459-463). The genes encoding human and rat Tudor-SN have been assigned to human chromosome band 7q31.3 and to rat chromosome band 4q23, respectively (Lienard et al., Cytogenet. Cell Genet., 2000, 90, 253-254).
In addition to the tudor and SN-like domains, Tudor-SN contains an EVES motif through which it interacts with the vertebrate transcriptional activator and proto-oncoprotein c-Myb (Dash et al., Genes Dev., 1996, 10, 1858-1869). Tudor-SN also interacts with the related transcription factors A-Myb and B-myb and serves to act as a coactivator and inhibitor of all three Myb proteins (Rushton et al., Blood Cells Mol. Dis., 2001, 27, 459-463). Tudor-SN also binds to the oncoprotein Pim-1, a serine/threonine kinase that is regulated by hematopoietic cytokine receptors and cooperates with c-myc in lymphoid cell transformation. Tudor-SN is phosphorylated by Pim-1 in vitro, and, in animal cells, forms a stable complex with Pim-1 and mediates the stimulation of c-Myb transcriptional activity by Pim-1 (Leverson et al., Mol. Cell, 1998, 2, 417-425). These data suggest that Tudor-SN serves to link the Myb proteins to common upstream signaling pathways.
U.S. Pat. No. 6,248,584, PCT publication WO 01/077168, and PCT publication WO 01/057058 are each incorporated herein by reference in their entirety.
Because RNAi has been demonstrated to suppress gene expression in adult animals, it is hoped that small non-coding RNA-mediated mechanisms might be used in novel therapeutic approaches such as attenuation of viral infection, cancer therapies (Shi, Trends in Genetics, 2003, 19, 9-12; Silva et al., Trends in Molecular Medicine, 2002, 8, 505-508).
Like the RNAse H pathway, the RNA interference pathway for modulation of gene expression is an effective means for modulating the levels of specific gene products and, thus, would be useful in a number of therapeutic, diagnostic, and research applications involving gene silencing. The present invention therefore provides oligomeric compounds useful for modulating Tudor-SN activity, including those relying on mechanisms of action such as RNA interference and dsRNA enzymes, as well as antisense and non-antisense mechanisms. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify additional oligonucleotide compounds for these uses.